Materials Science and Engineering C 24 (2004) 349 – 354

www.elsevier.com/locate/msec

Effect on liver cells of stepwise microstructures fabricated in a

photosensitive biodegradable polymer by softlithography
Eric Leclerc a, Fusae Miyata b, Katsuko S. Furukawa b, Takashi Ushida b,
Yasuyuki Sakai c, Teruo Fujii c,*
a
LIMMS/IIS-CNRS, University of Tokyo, 4-6-1 Komaba Meguro-ku Tokyo, 153-8505, Japan
b
Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo Bunkyo-ku Tokyo, 113-8654, Japan
c
Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba Meguro-ku Tokyo, 153-8505, Japan

Abstract

Two types of scaffolds composed of microstructured chambers made in a photosensitive biodegradable polymer were microfabricated by
softlithography techniques. The fabrication process is fast and flexible. Single- and multi-stepwise microstructures could be fabricated on one
polymer layer forming an array of microstructured chambers. The static cell culture of Hep G2 cells and fetal human hepatocyte (FHH) cells
were successfully performed on those microstructured layers and show the biocompatibility of this polymer. Morphological observations
have shown various tissue-like behaviours during the static cell culture of Hep G2 and FHH cells such as cells aggregations and 3D
arrangements. As a first physiological response of the cells to the stepwise structures, the albumin production rate has shown a higher cell
activity in the microstructured chambers compared to conventional flat dish cultures.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Tissue engineering; Photosensitive biodegradable polymer; Microfabrication

1. Introduction PLGA have been investigated. Characteristic scales of

Though numerous liver cell culture methods, like cul-
ture on collagen [1,2], co-culture of rat hepatocytes with
non-parenchymal liver cells [3], were developed so Far,
still, those methods do not lead to significant results [4].
The advance in microsystem technology has recently been
used to microfabricate various types of microbioreactors in
silicon or in biocompatible polymers to culture mammalian
cells [5 –9]. By adequate designing of microfluidic channel
networks in biocompatible polymers, those microbioreac-
tors have shown their efficiency and convenience to study
the cell behaviour in vitro [10]. With those microbioreac-
tors, it is interesting to perform some toxicity or cell
interaction studies. However, to achieve in vitro liver tissue
ready for implantation [11,12], those polymers are not
useful because those are not biodegradable.
To overcome this problem, techniques to fabricated
microstructures with biodegradable polymers such as
* Corresponding author. Fax: +81-3-5452-6213.
E-mail address: tfujii@iis.u-tokyo.ac.jp (T. Fujii).
the microstructures around 100 Am could be achieved by
softlithography techniques [5]. In this context, various
types of cell behaviours on biodegradable microstructures,
such as static culture of endothelial cells, were investigat-
ed [13]. To achieve microfluidic network for perfusion cell
culture, an original method of fabrication, based on a 3D
syringe deposition of PLLA, was developed. By this
method various complex three-dimensional networks could
be fabricated [14].
An alternative method for 3D fabrication is the photo
fabrication techniques. However, as conventional biode-
gradable polymers like PLGA and PLLA do not contain
required photosensitive group, new types of material must
be investigated. Following the research axis on fabrication
to get microbioreactors in biodegradable polymer for cell
culture in the frame of implantation applications, we
previously proposed softlithography based techniques to
fabricate various microstructured scaffolds using a new
type of photosensitive biodegradable polymer [15,16]. In
the present study, we will now investigate the effects of
the fabricated microstructures on liver cells. Static culture
of liver cells on the microfabricated structures with this

0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2003.12.004
350 E. Leclerc et al. / Materials Science and Engineering C 24 (2004) 349–354
Fig. 1. Hybrid fabrication process on stamping of microstructures
combining stamping and UV irradiation.
new type of polymer will be performed to investigate the
influence of the geometry and design on the cell behav-
iours. To do so, we used Hep G2 cells, human hepato-
carcinoma cells and fetal human hepatocytes (FHHs).
2. Material and methods
2.1. Material
A photosensitive polymer was used as a material of
fabrication of the microstructured scaffolds. The macro-
monomer, which is star-shaped poly-(q-caprolactone (CL)-
DL-lactide (LA)) tetraacrylate, was synthesized by a con-
ventional ring-opening reaction of LA using pentaerythiritol
as a initiator and by the addition of acryloil groups to the
end of the four CL-LA chains [15]. This type of photo-
cross-linkable macromonomer was first developed for drug-
delivery applications, but it has not been used in tissue
engineering. Preliminary evaluation of the polymer formed
by UV irradiation of the various types of synthesized
macromonomers showed that a macromonomer having a
Fig. 2. SEM views of (A) the single-stepwise microstructure and (B) the multi-stepwise microstructure fabricated with the present biodegradable polymer.
molecular weight of 5000 – 10,000 and a composition of
CL/LA = 50:50 gives the best characteristics (data not
shown). Namely, the macromonomer allows to get a soft
and elastic material after UV irradiation.
2.2. Fabrication techniques and microstructured scaffolds
design
The fabrication processes are previously described in
detail in the literature [16]. In the present study, two kinds
of microstructured scaffolds composed of microchambers
were prepared to achieve three-dimensional cell arrange-
ment. Both types of design are fabricated by a fabrication
process shown in Fig. 1. Using PDMS stamps, microcham-
bers were fabricated by transferring the desired pattern. A
scaffold with a single-stepwise structures was obtained as
shown in Fig. 2A. The microchambers in those scaffolds
have characteristic scales of 400400100 Am. Total
surface area, including the sidewall, was estimated to be
about 4.7 cm2, calculated for 900 microchambers. Then, a
scaffold of multi-stepwise structures was obtained by using
a multi-step PDMS stamp and scaffolds with four stepwise
polymer layers could be achieved as shown in Fig. 2B. The
dimensions of those chambers are shown in Fig. 2B and the
total surface is about 6.7 cm2, calculated for 324 micro-
chambers.
2.3. Cell culture and media
We used Hep G2, human hepatocarcinoma cells, as a
model liver cell [17]. The Hep G2 cells were obtained
from Japanese Collection of Research Bioresources
(JCRB). The culture medium for all the routine culture
and culture on microstructures was Dulbecco’s modified
Minimum Essential Medium (DMEM; Nissui Pharm.,
Tokyo, Japan) supplemented with 10% fetal bovine serum
(Filtron; Altona, Australia), 25 mM hydroxyethylpipera-
zine-NV2-ethanesulfonic acid (HEPES; Dojindo, Kuma-
moto, Japan), 100 units penicillin/ml (Wako), 100 Ag
streptomycin/ml (Wako) and 0.25 Ag amphotericin B/ml
(Sigma).
 E. Leclerc et al. / Materials Science and Engineering C 24 (2004) 349–354 351

Fetal human hepatocytes (FHHs) were originally iso-
lated by Applied Cell Biology Research Institute (Kirk-
land, WA, USA) from six fetal liver tissues by elutriation
followed by the digestion with dispase and distributed by
Dainippon Pharmaceutical (Osaka, Japan). The cells had
been subcultivated several times but the cumulative pop-
ulation doublings (CPD) were less than three times when
obtained, and we used them for the experiments in
additional five times of subcultivations (roughly additional
20 CPD) using the conventional trypsin/EDTA method.
The culture medium for the routine culture is DME/Ham
F-12 (1:1) (Life Technol., Rockville, MD, USA) supple-
mented with 10% FBS, 10 Ag recombinant human acidic
fibroblast growth factor (rh-aFGF)/ml (PeproTech EC,
London, UK), 25 Ag heparin/ml, 10 Ag gentamicin sulfate
(Wako) and 1 Ag amphotericin B/ml (Sigma). Dishes for
the culture were all coated with an acidic solution of
0.03% type I collagen (Nitta Gelatin, Osaka, Japan).
Expression of various liver-specific functions of FHHs is
reported to be very low even when compared to human
hepatoblastoma [18], Hep G2 cells in the recommended
serum-free culture medium, CS-C (Cell Systems, Kirkland,
WA, USA). Therefore, in the experiments with functional
assays, we used another new serum-free culture medium,
KSF (Yagai Research Institute, Yamagata, Japan), after
supplementation with 20 mM HEPES, 0.5 mM ascorbic
acid 2-phosphate (Wako), 10 Ag aFGF/ml, 10 Ag EGF/ml,
10 8 M glucagon (Sigma-Aldrich), 10 6 M hydrocortisone
(Wako), 25 Ag heparin/ml, 10 Ag gentamicin sulfate (Wako)
and 1 Ag amphotericin B/ml (Sigma). In this modified KSF
culture medium, growth of FHH cells was relatively sup-
pressed and their various liver specific functions were
enhanced. These results are submitted elsewhere [19].
Albumin production, which characterises the FHH and
Hep G2 cells activity, was measured by sampling periodi-
cally the culture medium. The measurement was done by a
sandwich-type enzyme-linked immunosorbent assay
(ELISA). Anti-human albumin goat-antibody and anti-hu-
man albumin goat-antibody conjugated with horseradish
peroxidase were purchased from Cappel Lab. (Ohio,

Fig. 3. Morphological observations of the cells during the culture on the single-stepwise microstructures of the present biodegradable polymer. Hep G2 cells on
the (A) 2nd, (B) 7th, and (C) 13th day. FHHs on the (D) 5th, (E) 7th, and (F) 12th day.
352 E. Leclerc et al. / Materials Science and Engineering C 24 (2004) 349–354

USA), and standard human albumin was purchased from assume that cells attached on the horizontal projected
Sigma Aldrich. surfaces of the stepwise microstructures.

2.4. Experimental 3. Results and discussions

After a sterilisation process performed with ethanol, the
scaffolds were put on a non-tissue-culture-treated dish
(bacterial culture grade). The dish and scaffolds were then
rinsed with water, phosphate-buffered saline solution (PBS)
and, finally, with an excess amount of culture medium. The
scaffolds were precoated with an acidic solution of 0.03%
type I collagen (Nitta Gelatin) for 30 min. Then, the cells
were inoculated on the scaffold and kept at rest overnight in
an incubator to enhance the attachment of the cells. On the
next day, the culture medium containing non-attached cells
was aspirated, and the scaffold was transferred into another
dish with the fresh culture medium. Then, the culture
medium was changed periodically once every 2 or 3 days
during the cell culture experiment.
Static cultures of Hep G2 cells and FHHs were con-
ducted on two types of polymer scaffolds, single-stepwise
and multi-stepwise microstructures, along with a tissue-
treated dish (10 cm2) as control. The experiments were
repeated at least three times. The initial cells density
inoculated was about 4104 cells/cm2 for all cases. We
3.1. Morphological observations
Microscopic observations showed good biocompatibility
of the biodegradable polymers, i.e., good initial cell attach-
ment, spreading, growth over the microstructures over the
entire duration of culture. Fig. 3A – C shows the Hep G2
cells situation on the 2nd, 7th and 13th day of the culture on
the single-stepwise microstructures. Cell growth and forma-
tion of a confluent layer was observed after 7 days. Several
spheroid-like cells reorganisation could be distinguished
after the confluent situation as shown in Fig. 3B. The
diameter of those spherical aggregates was estimated to be
about 200 Am. The aggregates were preferentially located
near the walls of microstructures and in the central part of
the microchambers. As shown in Fig. 3C, various three-
dimensional huge aggregates were observed and their con-
nections between several adjacent microchambers. Hep G2
cells cultured on dishes did not show any huge aggregates
which appears in the case of single-stepwise microstructures
as shown in Fig. 3C. With collagen coated on the biode-

Fig. 4. Albumin production during the culture of (A) Hep G2 cells and (B) FHHs in the present microstructures of biodegradable polymer. (n), single-stepwise
structure; (x), multi-stepwise structure; (.), tissue-culture-treated dish (control). Each point represents the mean F S.D. in three cultures.
 E. Leclerc et al. / Materials Science and Engineering C 24 (2004) 349–354
gradable polymer, the difference between the microstruc-
tured scaffold and the dish was the appearance of the
spheroidal cell arrangement and the huge aggregates con-
necting the microchambers.
Morphological observations of FHHs on the single-
stepwise microstructures are shown in Fig. 3D –F. As shown
in Fig. 3D and E, the cells attached and spread during the
first week of culture. Confluent morphologies were ob-
served forming a layer over the structures. After confluence,
large density of cell aggregates occurred after 1 week with a
characteristic scale of 100 Am as shown in Fig. 3F near the
walls of microstructure. Due to such three-dimensional
aggregates cells on the single-stepwise microstructures
could be connected to each other even if they are in the
different compartments (microchambers). In the case of
FHHs, any huge aggregates as in Hep G2 culture weren’t
observed. Compare to Hep G2, the difference might be
explained by higher attachability of the FHHs to the sub-
strates. The cells could be kept in good conditions about 2
weeks without subculture. In addition, during the dish
cultures such aggregates of FHHs were not observed even
when FHHs could be kept in over-confluent situation. This
illustrated once more the control of macroscale cell reor-
ganisation by the microscale topography of the single-
stepwise microstructures.
3.2. Albumin production
The measured values of daily albumin production in Hep
G2 and FHHs cultures on the microstructures of biodegrad-
able polymer are shown in Fig. 4A and B. For all experi-
ments, we observed that daily albumin production was
higher in the case of multi-stepwise microstructures than
that of the single-stepwise microstructures. Cellular activities
during the dish culture were found lower compared to those
of the culture done in single- and multi-stepwise micro-
structures. Both data, from FHHs and Hep G2 cells, appear
consistent with the positive effect of the microstructures. For
Hep G2 cells albumin production in the single- and multi-
stepwise microstructures reached after 12 days at around 60
and 90 Ag/day/106 cells, respectively. For FHHs albumin
production in both microstructures saturated after 9 days of
culture at around 1.25 and 2.25 Ag/day/106 cells for the
single- and multi-stepwise scaffolds, respectively.
4. Discussions
In the present paper, fast and reproducible fabrication
methods of single- and multi-stepwise microstructured scaf-
folds in photosensitive biodegradable polymer were pre-
sented. Then the scaffolds were used to perform some static
liver cells culture. Since those fabrication techniques are
flexible, they could be applied to fabricate and then test
several types of designs of single- and multi-stepwise
structured polymer layers. Comparing previous work done
353
on biodegradable polymers fabrication [13,16], this work
demonstrates the possibility of microfabrication on photo-
sensitive poly-caprolactone-lactide-based polymer for cell
culture scaffolds. The main advantage of this type of
photosensitive polymer is then the possibility to extend its
utilisation by using a more sophisticated facility like stereo-
lithography [20] which will allow to build more complicated
scaffolds for larger-scale cell culture, such that the fabricat-
ed scaffolds could be used in continuous perfusion culture.
On the effect of microstructures, an improvement of cell
activities on microstructured polymers was clearly obtained.
Spheroid aggregations of the Hep G2 cells or hepatocytes
3D structures were observed with characteristic sizes of
100 – 200 Am. In addition, cell’s interconnections between
microstructured chambers and layers could be observed as a
result of larger three-dimensional cell reorganisation. Cou-
pled to those morphological observations, the albumin
production of Hep G2 cells and FHHs was enhanced on
the single- and multi-stepwise microstructures.
Then, we could clearly observe an improvement of the
cell activities between the conventional dish culture and the
culture on the microstructures. The activities of Hep G2 cells
on the single-stepwise microstructures were five times higher
than those of the dish culture as shown in Fig. 4A, whereas
the surface area was smaller, namely 10cm2 and 4.4cm2 for
the single-stepwise microstructure and the dish, respectively.
The same behaviour was observed with the FHH cells in Fig.
4B, in which the albumin production was enhanced at least
by the factor of 10. Those quantitative results of the albumin
production of Hep G2 cells and FHH clearly have shown that
the effects of microstructured surfaces of the scaffolds are
significant in terms of cellular behaviour and activity. The
albumin production of the both types of cells are enhanced in
the cultures on microstructures.
On the contrary, the difference in albumin production
between the single- and multi-stepwise structures could be
explained by larger surface areas of culture involved in the
multi-stepwise culture. Due to the larger surface involved in
the culture, larger cell number is involved in the culture,
which explains the higher albumin production in the case of
multi-stepwise microstructures. Indeed for both types of
cells, the albumin production was enhanced by the factor
of 1.5 for the Hep G2 cells (see Fig. 4A) and 1.8 for the
FHHs (see Fig. 4B), whereas the surface area of the multi-
stepwise microstructures is 1.5 times larger than that of the
single-stepwise ones (see Section 2.2).
Considering those daily albumin productions for both
types of cells, maturation of FHHs in vitro is not enough
even in the case of multi-stepwise microstructures to attain
the levels of matured and adult cells. Albumindaily produc-
tion of the Hep G2 cells was about 50 times larger than that
of FHHs. This shows the necessity of further improvement
in culture condition for FHHs.
Larger multilayered cellular arrangement leading to
higher efficiency in albumin production could be estab-
lished in this work thanks to the present microfabrication
354 E. Leclerc et al. / Materials Science and Engineering C 24 (2004) 349–354
process with a photosensitive biodegradable polymer. This
behaviour looks consistent with the study done in 3D PLLA
random scaffolds in which the same behaviour was moni-
tored [19]. To achieve physiologically meaningful functions
of cells for liver implantation, it seems that a larger 3D
microfabricated structure arrangement in the bioreactor
might be required. Since the influence of 3D reorganization
of the cell was demonstrated [21] and appeared as a key
point for the maintenance of high functional activity in vitro,
the enhancement of the efficiency in the multi-stepwise
microstructures is consistent with the general understanding
of the behaviours of liver cells.
5. Conclusions
The photosensitive biodegradable polymer introduced in
this paper showed good compatibility with mammalian liver
cells. Single- and multi-stepwise microstructures were fab-
ricated and used in static cultures of Hep G2 cells and
FHHs. The cells have shown a good responses in terms of
arrangements and activity. The stepwise microstructures
have shown a contribution to enhance the albumin produc-
tion compare to conventional dish culture.
Acknowledgements
This work was performed in the framework of LIMMS, a
collaboration initiative between CNRS and The University
of Tokyo, with a support from JSPS. We thank M. Tsuru for
polymer synthesis and finally Dr. Kaoru Yoshimura, at
Musashino Chemical Laboratory, Ltd., for supplying us the
raw material DL-lactide.
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